- Demonstrated that rabbit antibodies targeting the HIV-1 gp41 heptad repeats can inhibit viral fusion and infection of primary cells. Use the same immunogens to isolate human monoclonal antibodies from a synthetic phage library expressing 1011 different single chain human antibodies (scFv) (collaborator: Carol Weiss) - Demonstrated that T. gondii cyclophilin A binds to human CCR5 and inhibits HIV-1 cell entry. - Used peptide-phage library to isolate a linear peptide mimicking the conformation-dependent epitope (mimetop) of the anti-CCR5 mAb 2D7, a potent inhibitor of HIV-1 infection. The peptide mimetop was used to generate 2D7-like antibodies in rabbits and to isolate 2D7-like single chain monoclonal antibodies (scFv) from a synthetic human scFv phage library. Both antibodies were shown to inhibit HIV-1 fusion in vitro. - Continued work on the development of heat-inactivated B. abortus as a bacterial delivery vector of HIV proteins, as a potential therapeutic vaccine for HIV infected patients (collaborator: Basil Golding, OBRR) - Developed a novel vaccinia neutralization assay based on the expression of the reporter gene b-galactosidase (b-Gal). This is a high throughput assay that is rapid (24 hr), sensitive (similar to the much slower classical plaque reductions assays), automated, and easily transferred to other laboratories. - The new vaccinia neutralization assay was used in multiple collaborative studies to evaluate vaccine take in recent smallpox vaccine recipients and to determine for how long titers are maintained in individuals who were vaccinated at childhood. - The new vaccinia neutralization assay was used to measure the potency of new Vaccinia immunoglobulin (VIG) products as well as IGIV products. - New animal models were established to measure vaccinia dissemination to internal organs and to measure the protective activity of VIG/IGIV in vivo, using protocols of post-exposure and prophylactic treatments against vaccinia. Evaluation of safety of monoclonal antibodies and polyclonal immunoglobulins against HIV and its cellular receptors. (1) Goals of project: - To study the activities of monoclonal antibodies (mAbs) or polyclonal antibodies raised against HIV-1 envelope (gp120), CD4, gp120/CD4 complexes, gp41-fusion intermediates, or the HIV-1 co-receptors, on HIV-1 fusion/infection of primary human cells. - To examine the effects of such mAbs on the normal biological functions of primary human cells. (2) Major Findings: - HIV-1 entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Sub-optimal temperature (31.5oC) was used to prolong fusion intermediates as monitored by confocal-microscopy. After transfer to 37oC, these fusion intermediates progressed to syncytia formation with enhanced kinetics compared with effector/target (E/T) cell mixtures that were incubated only at 37oC. gp41 peptides, DP-178 , DP-107, and IQN17, blocked fusion more efficiently (5-10 fold lower ID50 values) when added to E/T cells at the suboptimal temperature prior to transfer to 37oC. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37oC only if preincubated with E/T cells at the suboptimal temperature. Similar fusion-inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site. - New immunogens mimicking the gp41 fusion intermediates were constructed and several rabbits were immunized. The new rabbit sera inhibit HIV-1 fusion and infection of PBMC by several primary isolates when added to virus-cell mixtures at 37oC. - The same immunogens were used recently to isolate human monoclonal antibodies from a synthetic phage library expressing 1011 different single chain human antibodies (scFv) - Used peptide-phage library to isolate a linear peptide mimicking the conformation-dependent epitope (mimetop) of the anti-CCR5 mAb 2D7, a potent inhibitor of HIV-1 infection. The peptide mimetop was used to generate 2D7-like antibodies in rabbits and to isolate 2D7-like single chain monoclonal antibodies (scFv) from a synthetic human scFv phage library. Both antibodies were shown to inhibit HIV-1 fusion in vitro HIV-1 mediated membrane fusion as target for anti-viral therapy. (1) Goals of project: - To develop biological and biochemical assays for monitoring HIV-1 envelope-mediated cell fusion and formation of the tri-molecular complex between gp120/CD4/co-receptor. - To study the expression and function of HIV-1 co-receptors on primary human cells known to be targets for HIV-1 infection, and to study the effects of pro-inflammatory cytokines on the function of the HIV-1 co-receptors and infectivity of primary human cells. - Development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: - Biological (Ca++ flux, chemotaxis, HIV-fusion) and biochemical assays were developed to measure the association of the CD4/gp120 complex on human cells with the HIV-1 co-receptors for T-tropic and M-tropic envelopes (CXCR4 and CCR5). They included co-immunoprecipitations of CD4/co-receptors from cell lines and from primary human cells (i.e., monocytes and macrophages), and Western blots of whole cell and membrane extracts using our rabbit anti-CXCR4 and anti-CCR5 reagents. - Studies were conducted on multiple cell types: Langerhans cells (LC), dendritic cells (DC), Thymocyte subsets, CD34+ progenitors, peripheral blood T cells, monocytes (MO) and macrophages (MDM). - Develop new reagents for immunoprecipitation and Western blot analyses of the key HIV-1 co-receptors (CXCR4, CCR5). - Apply new biochemical assays including "Proteomics" to identifying the post-translational modifications of HIV-1 co-receptors that affect their function in primary human cells. (3) Major Findings: I. The chemokine receptor CXCR4 is a primary coreceptor for the HIV -1 virus. The predicted MW of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T lymphocyte line revealed multiple molecular weight isoforms of CXCR4. Three of the bands could either be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or co-precipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, anti-ubiquitin antibodies reacted with the 62 kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101 kDa CXCR4 isoform in immunoblots by three- to seven-fold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV -1 envelope-expressing cells. Similar increases in the intensity of the 101 kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Anti-ubiquitin antibodies reacted with multiple proteins above 62 kDa that were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for both proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary cells. Changes in expression and conformation of CXCR4 could affect the susceptibility of a given cell to infection by T-tropic HIV-1 strains, which only use CXCR4 as a coreceptor. Such changes may also indirectly influence infection by M-tropic HIV-1, which uses CCR5 as a coreceptor, since the two coreceptors compete with each other for interaction with CD4 (J. Virol. 74:5016-5023, 2000). In addition, changes in conformation may alter the effectiveness of vaccines and small drugs that target the coreceptors. Thus, it is crucial to understand the heterogeneity and functional diversity of CXCR4 and the other principal coreceptor, CCR5. We and others have recently observed a predominant CCR5 62 kDa species. Ubiquitination may play a role in the constitutive intracellular transport, turnover, and conformation of the HIV-1 coreceptors. Our earlier findings together with the current data suggest that mono-ubiquitination may enhance the role of CXCR4 and CCR5 as HIV-1 coreceptors by inducing constitutive association with CD4 in monocytes, perhaps by altering their conformation. On the other hand, polyubiquitination of CXCR4 may decrease its steady state surface expression in resting cells. These studies have been extended to compare unactivated and activated thymocytes and T cells. In a preliminary study we found that in human thymocytes and peripheral T cells changes in post-translational modifications as determined by changes in the predominant MW species in Western blots, are seen following cell activation. These changes correlated with enhanced fusion activity and increased association with CD4 molecules without a parallel increase in transcription or in the cell-surface density of the coreceptors. II. In studying the induction of cell mediated immunity to the protozoan Toxoplasma gondii we have discovered that a protein, C-18 (cyclophilin) from the parasite binds to both murine and human CCR5. In vitro studies demonstrated that recombinant C-18 is effective in neutralizing infectivity of both lab-adapted and multiple primary HIV R5-isolates from different clades but was inactive against X4 viruses. Unlike previously described chemokine analogs used as co-receptor antagonists, C-18 does not appear to trigger receptor internalization and as foreign molecule is unlikely to induce autoimmunity. It therefore could provide the basis for the development of a new microbially derived antagonist lacking these deleterious side effects. Evaluation of new carriers and adjuvants for HIV-1 vaccines. (1) Goals of Project: - To identify a carrier which will increase the immunogenicity of the HIV-1 subunits, and will be able to recall anti-HIV B memory cells in patients with pre- existing immune deficiency. - To identify a carrier which would augment the TH1/TH2 ratio of infected individuals and will favor generation of cellular responses including cytotxic cells (CTL) and beta chemokines production. (2) Experimental approaches: - The gram negative Brucella abortus (Ba), and LPS derived from its cell wall (Ba- LPS), were tested as carriers for either inactivated HIV-1 virions, gp120 (SF2) glycoprotein, or peptide derived from the V3-loop of HIV-1 (MN) env. The different conjugates were used to immunize mice with different degrees of T cell deficiency. Four Rhesus macaques were also vaccinated with a Ba-V3 conjugate. - Both humoral and cytotxic immune responses were measured including systemic and mucosal antibody responses. Biologically relevant antibodies were measured in syncytia inhibition assays. - In vitro studies with human T cells and elutriated monocytes from normal as well as HIV-1 infected patients were assessed for their lymphokine production in response to Ba and Ba-LPS by PCR and biological assays. - PCR primers were designed to test the ability of Ba, Ba-LPS,and Ba/DNA, to elicit chemokines from human PBL (3) Major findings: - Ba conjugated to a peptide containing B-cell epitope and CTL epitope (N3v3), generated both neutralizing antibodies and cytotoxic T cells capable of killing HIV-infected targets. CD4 - depleted mice retained their ability to generate anti-HIV neutralizing Ab and CTL after immunization with the Ba-N3V3 conjugate. - Ba and Ba-LPS were found to directly activate purified human CD4-positive TH1 cells, and to a lesser degree, CD8-positive cells, as judged by induction of the lymphokines IL2 and IFN-gamma. PBL from HIV-1 infected individuals were also responsive. Ba and Ba-LPS can also activate IL12 secretion by human monocytes. - Rhesus macacques produced high titer HIV-1-neutralizing antibodies in the serum and mucosal surfaces.In addition,IFNgama producing cells and beta-chemokine producing cells were measured after Ba-gp120 immunization. - Inactivated Ba , as well as Ba derived DNA and LPS were found to induce mRNA for RANTES, MIP-1a, and MIP-1b in human PBL (in 20 hr) , and secretion of these chemokines from CD8+ and CD4+ cells and from monocytes. These chemokines will add to the anti-viral millieu in vaccinated individuals. The beta chemokines can block and prevent infection of R5 viruses by binding and/or downregulation of the CCR5 HIV-1 coreceptor. - new efforts are underway to clone HIV-1 sructural genes (Gag/Pol) into plasmid and infect attenuated strain of Ba, used as a veterenary vaccine. The bacterium containing the recombinant plasmid will be killed by heat and will be tested for generation of CTL in BALB/c mice and rhesus macaques.So far we were successful in expressing the HIV pol gene in two plasmids that were expressed in transfected attenuated Brucella abortus strain RB51 Development of new in vitro assays for the assessment of vaccinia virus neutralization. With the potential threat of smallpox as a bioterrorism agent, a major effort should be invested in two main areas: a) development of new effective preventive vaccines, and b) production of high titer vaccinia IGG (VIG) that can be used as a first line of defense in exposed and/or high risk populations. The current stocks of VIG are limited and outdated. Scientists at CBER and elsewhere (MPH) were encouraged to produce new lots of VIG and subject them to careful titration in validated neutralization assays. In addition, experiments are underway to compare the neutralization capacity of different VIG subclasses, in order to optimize viral neutralization in future production of VIG.The current neutralization assay for vaccinia (plaque-reduction), is labor intensive, slow (up to 7 days), and depends on scoring plaques by eye (i.e., subjective). This assay is difficult to validate and to transfer to sponsor-based laboratories. As an alternative approach, our laboratory initiated late last year the development of an alternative vaccinia neutralization assay, which is fast, quantitative, and easy to perform and transfer to other laboratories.This assay is based on the use of recombinant vaccinia virus expressing a bacterial gene coding for the enzyme b-Gal under the control of the vaccinia late promoter. Results: - We have established and standardized a novel assay to measure vaccinia neutralization. It is based on the expression of a reporter gene coding for the bacterial b-galactosidase enzyme (b-Gal) under the control of a synthetic E/L promoter. We demonstrate that the new assay is rapid (24 hr), of equal sensitivity to PRNT assays, reproducible, objective, and easy to transfer. In addition, we describe preliminary results with a second reporter gene assay based on a vaccinia-EGFP recombinant virus. The expression of the GFP is induced by IPTG. The two assays provide high throughput capabilities and may be established in clinical laboratories for the evaluation of multiple samples from clinical trials. - Thus far, it was found that the MPH VIG is somewhat better than the Baxter (4oC) VIG with ID(50) values of 10-15 ug/ml and 25 ug/ml, respectively.The new VIG product was found to be superior to the old VIG products. - We tested multiple lots of IVIG from 5 different manufacturers. Several lots were found to have significant anti-vaccinia neutralization titers. - The new vaccinia neutralization assay was transferred to several laboratories involved in new product evaluation and vaccine trials (new smallpox vaccines). - The new vaccinia neutralization assay was also used in collaborative studies to evaluate the safety and efficacy of more attenuated smallpox vaccines designed for immunodefficient individuals, using primates infected with SIV (collaborator: Veffa Franchini, NCI/NIH) Evaluation of vaccinia IgG (VIG) in the protection of mice from vaccinia infection. The goal of the current project is to conduct an animal protection study with several VIG preparations in order to validate the in vitro neutralization assay. The results will greatly enhance the ability of CBER to streamline the testing of old and new VIG products and to alleviate the current shortage of VIG. These efforts will enhance CBER contributions to the Bioterrorism prevention efforts and will promote the testing of new recombinant vaccinia-based vaccines against HIV-1 and cancer. The specific goals are: Stage 1: To establish the optimal dose of vaccinia strains vSC56 and Wyeth that can be used to validate our in vitro viral neutralization assay. We expect death to occur within 10-20 days. Stage 2 and 3: To test multiple preparations of Vaccinia Immunoglobulins (VIG) and determine their IC50. This will involve determining the concentration of VIG required to protect 50 percent of the animals from a lethal dose of vaccinia virus infection. Results: - SCID mice were found to be more susceptible to vaccinia infection than nude mice.The immunodeficiency of SCID mice is more severe than that of the nude mice. - Both strains of vaccinia that were tested, Wyeth vaccine strain and vSC56, killled SCID mice between 25-35 days post infection (10^6 pfu/mouse, IP). - In the first in vivo mouse protection study, Baxter and MPH VIG were incubated with Wyeth vaccinia virus, either undiluted or at 1:10 and 1:100 dilutions of the VIG preparations. - Undiluted MPH VIG was completely protective. 1:10 and 1:100 dilutions were partially protective. They postponed animal death by 1-2 weeks. - The Baxter VIG (4oC lot) was less protective than the MPH. one death was observed with undiluted preparation and 1:10 VIG dilution. The 1:100 dilution was not significantly protective. - Since the concentration of IgG in Baxter VIG is >3 fold higher than in the MPH preparation, our preliminary results suggest that the MPH preparation should be the product of choice in the case of smallpox exposure or in the case of viremia associated with recombinant vaccinia vector vaccinations. On going studies: - Repeat of the first study using the Wyeth vaccinia virus and multiple dilutions of Baxter and MPH VIG preparations (1:4 serial dilutions). - Similar protection study using vSC56 virus (the virus strain which is used in the in vitro neutralization assays). - We recently developed new animal model to study vaccinia dissemination to internal organs following inoculations via multiple routes: intraperitoneal, intranasal, subcutaneous, and intradermal. We are using the vSC56 and measure b-Gal activity in multiple organs (spleen, liver, heart, brain, lungs, kidneys, ovaries, and testes) at multiple time points. - The new in vivo animal models (in normal or immunodeficient mouse strains) are used to evaluate the safety of the current smallpox vaccine (Dryvax) and the efficacy of VIG and monoclonal antibodies in protecting animals from adverse events and/or lethality This project incorporates FY2002 projects 1Z01BK003002-10, 1Z01BK003003-10, 1Z01BK003004-10, 1Z01BK003019-02, and 1Z01BK003020-02.